Apparatus and methods for microbial identification by mass spectrometry

ABSTRACT

Methods and systems for identification of microorganisms either after isolation from a culture or directly from a sample. The methods and systems are configured to identify microorganisms based on the characterization of proteins of the microorganisms via high-resolution/mass accuracy single-stage (MS) or multi-stage (MS n ) mass spectrometry. Included herein are also discussion of targeted detection and evaluation of virulence factors, antibiotic resistance markers, antibiotic susceptibility markers, typing, or other characteristics using a method applicable to substantially all microorganisms and high-resolution/mass accuracy single-stage (MS) or multi-stage (MS n ) mass spectrometry.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Prov. Pat.App. Ser. No. 61/687,785 entitled “Apparatus and methods for microbialanalysis and directed empiric therapy” filed 1 May 2012 with inventorsJames Stephenson, Oksana Gvozdyak, Roger Grist, Clay Campbell and IanJardine, the entirety of which is incorporated herein by reference.

BACKGROUND

In recent years, mass spectrometry has gained popularity as a tool foridentifying microorganisms due to its increased accuracy and shortenedtime-to-result when compared to traditional methods for identifyingmicroorganisms. To date, the most common mass spectrometry method usedfor microbial identification is matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass spectrometry. In MALDI-TOF,cells of an unknown microorganism are mixed with a suitable ultravioletlight absorbing matrix solution and are allowed to dry on a sampleplate. Alternatively, extract of microbial cells is used instead of theintact cells. After transfer to the ion source of a mass spectrometer, alaser beam is directed to the sample for desorption and ionization ofthe proteins and time-dependent mass spectral data is collected.

The mass spectrum of a microorganism produced by MALDI-TOF methodsreveals a number of peaks from intact peptides, proteins, and proteinfragments that constitute the microorganism's “fingerprint”. This methodrelies on the pattern matching of the peaks profile in the mass spectrumof an unknown microorganism to a reference database comprising acollection of spectra for known microorganisms obtained usingsubstantially the same experimental conditions. The better the matchbetween the spectrum of the isolated microorganism and a spectrum in thereference database, the higher the confidence level in identification ofthe organism at the genus, species, or in some cases, subspecies level.Because the method relies upon matching the patterns of peaks inMALDI-TOF mass spectra, there is no requirement to identify or otherwisecharacterize the proteins represented in the spectrum of the unknownmicroorganism in order to identify it.

Although MALDI-TOF methods are rapid and cost effective, they havelimitations that restrict the range of applications. The informationcontent within a MALDI mass spectrum reflects the most abundant andionizable proteins which, except for viral, are generally limited toribosomal proteins at the experimental conditions used. Becauseribosomal proteins are highly conserved among prokaryotes,differentiation of closely related microorganisms by MALDI-TOF islimited. Moreover, determination of strain and/or serovar type,antibiotic resistance, antibiotic susceptibility, virulence or otherimportant characteristics relies upon the detection of protein markersother than ribosomal proteins which further limits the application ofMALDI-TOF for microbial analysis. Laboratories using MALDI-TOF foridentification of microorganisms must use other methods to furthercharacterize the identified microbes. In addition, the MALDI-TOFmethod's reliance upon matching spectral patterns requires a pureculture for high quality results and is not generally suitable fordirect testing of samples containing different microorganisms.

Several other mass spectrometry methods for detection of microorganismshave been used. For example, mass spectrometry-based protein sequencingmethods have been described wherein liquid chromatography is coupled totandem mass spectrometry (LC-MS/MS) and sequence information is obtainedfrom enzymatic digests of proteins derived from the microbial sample.This approach, termed “bottom-up” proteomics, is a widely practicedmethod for protein identification. The method can provide identificationto the subspecies or strain level as chromatographic separation allowsthe detection of additional proteins other than just ribosomal proteins,including those useful for characterization of antibiotic resistancemarkers and virulence factors. The main drawback of the bottom-upapproach is the extended time to result due to the need for proteindigestion, long chromatographic separation and data processing time.Therefore, this method is not amenable to high throughput approaches.

BRIEF SUMMARY

The present invention includes a novel method and system foridentification of microorganisms either after isolation from a cultureor directly from a sample based on the characterization of proteins ofthe microorganisms via high-resolution/mass accuracy single-stage (MS)or multi-stage (MS^(n)) mass spectrometry. Included herein are alsodiscussion of targeted detection and evaluation of virulence factors,antibiotic resistance markers, antibiotic susceptibility markers, orother characteristics using a methodology applicable to substantiallyall microorganisms and high-resolution/mass accuracy single-stage (MS)or multi-stage (MS^(n)) mass spectrometry. And while the followingdiscussion focuses on the identification of microorganisms via thecharacterization of proteins, the methods and systems discussed hereinare equally applicable to the identification of microorganisms via thecharacterization of one or more of small molecules, lipids, orcarbohydrates, and the like.

The present invention, in one aspect, offers an alternative totraditional bottom-up proteomics methods, namely top-down analysis ofintact proteins derived from microbial cells via a method which isapplicable to substantially all microorganisms including Gram positivebacteria, Gram negative bacteria, mycobacteria, mycoplasma, yeasts,viruses, and filamentous (i.e., microscopic) fungi. The presentinvention provides identification of microorganisms at the genus,species, subspecies, strain pathovar, and serovar level even in samplescontaining mixtures of microorganisms and/or microorganisms analyzeddirectly from pure and/or mixed cultures and from direct samples (e.g.,surface swabs, bodily fluids, etc.). In addition, the approach can beemployed for targeted detection of virulence factors, antibioticresistance and susceptibility markers or other characteristics. Themethod of the present invention is simple and quick because there is noneed for chemical or enzymatic digestion of a sample and data processingis accomplished in real time.

An exemplary method involves a two-phase process. In the first phase,soluble proteins from microbes present in a sample are quickly extractedand analyzed with a mass spectrometer to identify the microbes basedupon molecular weight value and fragmentation analysis determined forone or more of the extracted soluble proteins. This first phase isperformed within a few minutes, for example, less than 10 minutes, lessthan 5 minutes or within about one minute or less. The second phaseutilizes rapid chromatographic separation and mass spectral analysis(e.g., targeted MS and MS^(n)) to further characterize the microbesidentified in the first step, for example, by determining virulencefactors, antibiotic resistance markers, antibiotic susceptibilitymarkers or other characteristics. This second phase is performed inwithin a few minutes, for example, less than 15 minutes, less than 10minutes or within about five minutes or less. Both phases rely on thedetection and identification of intact proteins derived from themicrobes, without chemical, physical or enzymatic degradation of thoseproteins to their substituent peptides.

Another exemplary method for identifying and characterizing one or moremicrobes in a sample includes steps of (a) performing a first analyticalmethod using mass spectrometry to detect and identify one or more (e.g.,one, two, three, four, five, or more) proteins from each of the one ormore microbes, (b) using the identity of the one or more proteins fromeach of the one or more microbes to further identify at least one of themicrobes in the sample, (c) using information from step (b) toautomatically select a second analytical method from a list ofpre-defined analytical methods, the second method also using massspectrometry, and (d) performing the second analytical method on thesample to determine if proteins indicative of antibiotic resistancemarkers, antibiotic susceptibility markers and/or virulence factors arepresent in the sample and, optionally, quantifying the antibioticresistance markers, antibiotic susceptibility markers and/or virulencefactors present in the sample.

Target microorganisms include, without limitation, Gram-positivebacteria, Gram-negative bacteria, mycobacteria, mycoplasma, viruses,yeasts and filamentous fungi. The characterization process may includethe detection of virulence factors, resistance markers, antibioticsusceptibility and any other molecules produced by the organisms ofinterest including, without limitation, those that impact clinicaloutcome. The method is applicable to a variety of different sampletypes, including samples from pure or mixed culture derived fromclinical samples including, without limitation, blood, urine, stool,sputum, wound and body site swabs, and to samples derived from othersources including industrial or environmental samples such as food,beverage, soil, water, air, and swabs of surfaces.

The method of the present invention comprises at least one or more ofthe following steps: microbial cell disruption, solubilization ofproteins, sample clean-up (to desalt, remove insoluble components anddebris, and/or concentrate), sample infusion or flow injection, fastpartial liquid chromatographic separation, ionization of proteins insolution, high-resolution/mass accuracy multi-stage mass spectrometry inMS and MS/MS mode, and microbial identification via molecular weightanalysis and/or protein sequence analysis.

The system and sample preparation kits of the present invention providemeans for performing the method. As contemplated in one embodiment, arapid extraction procedure is followed by on-line clean up and directanalysis. In another embodiment, rapid extraction is followed by fastpartial liquid chromatographic separation of intact proteins. Theproteins are then ionized, for example, via electrospray ionization. Theintact proteins are analyzed via MS and MS^(n) in order to identify themicroorganism to the genus, species, strain, subspecies, pathovar orserovar level, as needed. The MS or MS^(n) methods can employ directsequencing or pattern matching approaches for pathogen identification.The identification process occurs in real-time during the acquisitionperiod. It can occur post-acquisition as well. The system furtherprovides for quantitative detection and identification of virulencefactors, resistance markers, antibiotic susceptibility markers and/orany other relevant markers, for example, those associated with disease.

Because a common method, using a limited set of reagents, is performed,the method of the present invention is suitable for use within acompletely automated system for sample preparation and massspectrometry.

Ideally, the method of the present invention is automated from samplepreparation through results reporting. Results may be automaticallytransferred to a hospital's electronic medical records system where theycan be directly linked to patient treatment strategies, insurance,billing, or used in epidemiological reporting. Such an integrated systemfacilitates epidemiological tracking of an outbreak at the hospital,local, regional, and global levels. For high throughput laboratories,multiple systems can be interfaced to a central computer whichintegrates data from the different instruments prior to reporting. Thesystem can import phenotypic susceptibility data where it can becombined with identification, virulence, antibiotic resistance andtyping information generated by the invention.

These and other objects and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent disclosure, a more particular description of the disclosure willbe rendered by reference to specific embodiments thereof, which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the disclosure and aretherefore not to be considered limiting of its scope. The disclosurewill be described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1A is a flow diagram illustrating a method for identifying amicroorganism;

FIG. 1B is a flow diagram schematically illustrating an algorithm foridentifying a microorganism;

FIG. 2 is a block diagram schematically illustrating a system for rapidextraction and analysis of soluble proteins from at least onemicroorganism for identifying the at least one microorganism;

FIG. 3 is a diagram schematically illustrating a flow path that can beused according to one embodiment in the system illustrated in FIG. 2;

FIG. 4 illustrates a full scan electrospray mass spectrum of an E. coliextract performed via direct infusion;

FIG. 5 illustrates a mass isolation of a 50 Da window of the E. coliextract shown in FIG. 4;

FIG. 6 illustrates tandem mass spectrometry of the 50 Da windowillustrated in FIG. 5;

FIG. 7 illustrates an MS/MS fragmentation of the +19 charge state of DNABinding Protein H-sn from E. coli;

FIGS. 8A-8E illustrate mass spectrometry data for fast partialchromatographic separation of soluble proteins extracted from Bacilluslicheniformis, Candida albicans, Kocuria rosea, Staphylococcus xylosus,and Mycobacterium smegmatis;

FIG. 9A shows MS data illustrating a comparison of an antibioticresistant E. coli (ATCC 35218) grown under standard growth conditionsand in the presence of oxacillin for an 18 hr period;

FIG. 9B shows MS data illustrating a comparison of the antibioticresistant E. coli of FIG. 9A grown under standard growth conditions andin the presence of naficillin for an 18 hr period;

FIG. 9C shows MS data illustrating a comparison of the antibioticresistant E. coli of FIG. 9A grown under standard growth conditions andin the presence of penicillin for an 18 hr period;

FIG. 9D shows MS data illustrating a comparison of the antibioticresistant E. coli of FIG. 9A grown under standard growth conditions andin the presence of ampicillin for an 18 hr period;

FIG. 10 illustrates high resolution/mass accuracy extracted ion profilesfrom four different proteins derived from C. albicans;

FIGS. 11A-11C illustrate FPCS-MS total ion current profiles of variousmicroorganisms grown on the Oxoid Tryptic Soy Agar at 34 C for 20 h.(A)—Escherichia coli ATCC 8739; (B)—Enterococcus gallinarum ATCC 700425;(C)—Bacillus subtilis subs. spizizenii ATCC 6633; and

FIG. 12 illustrates deconvoluted masses of proteins derived from theinformation obtained in FPCS-MS of Escherichia coli ATCC 8739 grown onthe Oxoid Tryptic Soy Agar at 34 C for 20 h shown in FIG. 11A

DETAILED DESCRIPTION

The present invention, in one embodiment, provides a method for rapidextraction and analysis of a soluble protein extract from cells of atleast one microorganism, including Gram positive bacteria, Gram negativebacteria, yeasts, mycobacteria, mycoplasma, microscopic fungi, andviruses. Analysis of proteins is performed via mass spectrometry toidentify the microorganisms present in the sample and then, optionally,a targeted mass spectrometric analysis may be conducted to characterize(qualitatively and quantitatively) proteins associated with antibioticresistance and/or sensitivity markers, virulence factors, typing ofstrains, or other characteristics. In another embodiment, kitscomprising two or more of: reagents, consumables, devices, calibrators,controls, and standards for performing the method are provided.

FIG. 1A provides an overview of the general work flow of the method 100for rapid extraction and analysis of a soluble protein extract fromcells of at least one microorganism. The steps of the method 100 may beperformed manually using a variety of independent instruments anddevices. Alternatively, some or all of the steps may be automated. Anexemplary automated system suitable for performing the method 100 ofFIG. 1A is illustrated in FIG. 2. Further discussion of the exemplaryautomated system may be found in WO 2012/058632 and WO 2012/058559, theentireties of which are incorporated herein by reference.

Referring now to FIG. 2, a system 200 for extraction of proteins fromone or more microorganisms, detection of the proteins, andidentification of the one or more microorganisms is schematicallyillustrated. The system 200 includes a sample handling device 215, asample 210 that is accessible by the sample handling device 215, andreagents, buffers, and the like 220 that are fluidly coupled to thesample handling device 215. The system 200 further includes first andsecond solid phase extraction devices 235 (e.g., a solid phaseextraction cartridge) configured for cleaning up (e.g., desalting,removing contaminants, concentrating proteins) and an optionalchromatography column 240 that may be configured for at least partiallypurifying a sample 210 by liquid chromatography prior to mass-specanalysis. The sample 210, the first and second extraction devices 235,and the optional chromatography column 240 are in fluid communicationwith a fluid handling pump 230, the reagent 220, and a mass spectrometer250.

The sample handling device 215 is capable of preparing a range of sampletypes containing one or more microbes and delivering a soluble proteinfraction extracted from the microbes to the mass spectrometer 250 foranalysis. A sample 210 may be of any type suspected to contain one ormore microorganisms including, without limitation, isolated coloniesfrom a culture plate, cells from liquid growth medium, blood, saliva,urine, stool, sputum, wound and body site swabs, soil, food, beverage,water, air, and environmental surface swabs.

The sample handling device 215 may include one or more of a celldisruption means, a robotic liquid handling means, a centrifuge,filtration means, an incubator, mixing means, a vacuum pump, a fluidpump, and reagents 220 that can be used for disruption of microbes andisolation of a soluble protein fraction. Disruption of bacterial,fungal, mycoplasma cells, viruses, and the like may be achieved bymechanical, chemical, enzymatic and other means as are commonly known inthe art. Mechanical approaches include bead beating, use of pressurelike French press and the like, sonication or other methods known in theart. Chemical methods include exposure to chaotropes such as urea,thiourea, or guanidine HCL to lyse the microbial cells and solubilizetheir contents. Alternatively, organic acid/solvents mixtures may beutilized to disrupt cells. Enzymatic methods include using lysozyme,lysostaphin or other lytic enzymes to form “holes” in the bacterial cellwalls that allow the contents to leak out into the surrounding solution.

As illustrated in FIG. 2, the system 200 further includes an optionalcontrol unit 260 that can be linked to various components of the system200 through linkages 270 a-270 d. For example, the control unit 260 canbe linked to the sample 210 to control sample application, the reagents220 to control the application of various reagents, the pump 230 tocontrol fluid handling, flow rates, etc., to the sample handling device215 to control sample preparation, and to the mass spectrometer 250 tocontrol mass spectrometry parameters. In the illustrated embodiment, thecontrol unit 260 can also serve as a data processing unit to, forexample, process data from the mass spectrometer 250 or to forward thedata to server(s) for processing and storage (the server is not shown inFIG. 2), The Control Unit 260 can also be used to automatically forwardthe results to health care professionals.

In some embodiments, the system 200 is designed to be used by aclinician or a general laboratory technician who is not necessarilyexpert in all aspects of sample preparation, LC-MS operations, LC-MSmethods development, and the like. As such, the control unit 260 can bedesigned to encapsulate the data system environment by providing a userwith a simplified application interface that can be used to initiate andmonitor essentially all aspects of assaying a sample 210 withoutrequiring the user to interact with the overall hardware and controlsystems of the system 200. The control unit 260 is therefore configuredto provide a degree of separation between the user and the underlyingservices that control devices, data files and algorithms for translatingdata to a user readable form. That is, the control unit 260 eliminatesthe need for the user to be aware of or in control of hardware foranalyzing clinical samples and provides a simplified interface to sendand receive information from the mass spectrometer.

The control unit 260 may be configured to internally monitor each sampleanalysis request and is capable of tracking the analysis request fromstart to finish through the system 200. Once data for a sample 210 isbeing acquired or has been acquired by the system 200, the control unit260 may be configured to automatically start post processing the databased on the type of assay selected by the user. Moreover, the controlunit 260 can be configured to automatically select post processingparameters based on the type of assay selected by the user, furtherreducing the need for the user to interact with the system once theassay has been selected and started for analysis. The control unit 260can be designed as a layer that fits between the system 200 and the userto reduce the complexity needed to set up sample assays for acquisition.The control system 260 can also be configured to return only the mostrelevant data to the user to avoid overwhelming the user with extraneousinformation.

In one embodiment, the system 200 can further include a sample detectiondevice (not pictured) operably coupled to or integrated with the samplehandling device 215. The sample detection device can work with thesample handling device 215 or independently of the sample handlingdevice 215 perform at least one of the following functions:

-   -   i. identify samples entering the system;    -   ii. identify assay types for the samples entering the system;    -   iii. select an assay protocol based on the anticipated assay        type and/or analyte of interest;    -   iv. direct the sample handling device and/or the control system        to initiate analysis of the analyte of interest in the sample;    -   v. direct the control system to select one or more reagents        based upon the assay protocol selected for the type of assay        and/or analyte of interest;    -   vi. direct the control system to select a liquid chromatography        mobile phase condition based upon the assay protocol selected        for the type of assay and/or analyte of interest and cause the        liquid chromatography system to perform the assay and/or purify        the analyte of interest;    -   vii. direct the control system to select a mass spectrometer        setting based upon the assay protocol selected for the assay        type and/or analyte of interest and cause the mass spectrometer        to create mass spectral data associated with the selected assay        type and/or analyte of interest; or    -   viii. direct the control system to analyze the mass spectral        data associated with the selected assay type and/or analyte of        interest to identify the presence and/or concentration of the        analyte of interest.

The sample, or the processed sample, may be cleaned up and or purifiedprior to analysis by mass spectrometry. Such purification, or sampleclean-up, may refer to a procedure that removes salts or lipids from thecrude cell extract, or to a procedure that enriches one or more analytesof interest relative to one or more other components of the sample. Inone embodiment, such purification, or sample clean-up, may beaccomplished by the protein extraction devices 235 and/or the optionalchromatography column 240.

In one embodiment, the first and/or second extraction device 235 mayinclude a solid phase extraction (SPE) cartridge. In some embodiments,the SPE cartridge 235 may be in line directly with the highresolution/high mass accuracy mass spectrometer 250. In one embodiment,the SPE cartridge may be a polypropylene tip with a small volume ofsilica or other sorbent containing bonded C₄, C₈ or C₁₈ or otherfunctional groups immobilized in the cartridge, for example, a StageTip™cartridge (Thermo Fisher Scientific). In alternative embodiments,polymeric sorbents or chelating agents may be used. The bed volume maybe as small as 1 μL or less but greater volumes may also be used. Theapparatus and method are well suited to the complex samples derived fromthe microbial cells because each SPE cartridge is used only once,minimizing carryover problems from one sample to another.

In one embodiment, the optional chromatography column 240 may includecolumn configured for at least partial chromatographic separation of theproteins in the sample. The stationary phase in the chromatographycolumn may be porous or non-porous silica or agarose particles, or amonolithic material polymerized or otherwise formed inside the column.The stationary phase may be coated with an appropriate material such asC₁₈, C₈, C₄ or another suitable derivative, or contain cation exchangeror other material, or the combination of the above to facilitate theseparation of the proteins, and such material may be chemically bondedto the particles or monolith inside the column. Particle sizes typicallyrange from about 1.5 to 30 μm. Pore sizes can range from 50 to 300angstroms. Inside diameters of columns typically range from about 50 μmto 2.1 mm, and column length from about 0.5 cm to 25 cm, or other. Themobile phase or eluent may be a pure solvent, or a mixture of two ormore solvents, and may contain added salts, acids and/or other chemicalmodifiers. The proteins are separated on the column based on one or morephysiochemical properties, including size, net charge, hydrophobicity,affinity, or other physiochemical properties. Chromatographic separationmethods include one or more of ion exchange, size exclusion, HILIC,hydrophobic interaction, affinity, normal-phase, or reverse-phasechromatography.

Additional methods of purifying the samples may include, withoutlimitation, liquid chromatography, HPLC, UHPLC, precipitation,solid-phase extraction, liquid-liquid extraction, dialysis, affinitycapture, electrophoresis, filtration, ultrafiltration or other suitablemethods known in the art, are used for the purification.

Various methods have been described involving the use of HPLC for sampleclean-up prior to mass spectrometry analysis. One of skill in the artcan select HPLC instruments and columns that are suitable for use in theinvention. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties inspace and time. The medium may include minute particles. The particlesmay include a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the analytes of interest. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded surface. Alkyl bonded surfaces may include C₄, C₈, or C₁₈ bondedalkyl groups, preferably C₁₈ bonded groups. The chromatographic columnincludes an inlet port for receiving a sample and an outlet port fordischarging an effluent that includes the fractionated sample. Forexample, a test sample may be applied to the column at the inlet port,eluted with a solvent or solvent mixture, and discharged at the outletport. In another example, more than one column may be used sequentiallyor as a 2D chromatography wherein a test sample may be applied to afirst column at the inlet port, eluted with a solvent or solvent mixtureonto a second column, and eluted with a solvent or solvent mixture fromthe second column to the outlet port. Different solvent modes may beselected for eluting the analytes. For example, liquid chromatographymay be performed using a gradient mode, an isocratic mode, or apolytyptic (i.e. mixed) mode.

The terms “mass spectrometry” or “MS” as used herein refer to methods offiltering, trapping, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z” (also sometime referred to as “Da/e”). Ingeneral, one or more molecules of interest, such as microbial proteins,are ionized and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of electric ormagnetic and electric fields, the ions follow a path in space that isdependent upon mass (“m” or “Da”) and charge (“z” or “e”).

The mass spectrometer 250 will include an ion source for ionizing thefractionated or not fractionated sample and creating charged moleculesfor further analysis. For example ionization of the sample may beperformed by electrospray ionization (ESI). Other ionization techniquesinclude, but are not limited to, atmospheric pressure chemicalionization (ACPI), photo-ionization, electron ionization (EI), chemicalionization (CI), fast atom bombardment (FAB)/liquid secondary ion massspectrometry (LSIMS), matrix-assisted laser desorption ionization(MALDI), field ionization, field desorption, thermospray/plasmasprayionization, and particle beam ionization. The skilled artisan willunderstand that the choice of ionization method can be determined basedon the analyte to be measured, type of sample, the type of detector, thechoice of positive versus negative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z) and signal intensity. Suitableanalyzers for determining mass-to-charge ratios include quadrupoleanalyzers, ion trap analyzers, Fourier transform ion cyclotron resonance(FTICR) analyzers, electrostatic trap analyzers, magnetic sectoranalyzers and time-of-flight analyzers. The ions may be detected byusing several detection modes. For example, selected ions may bedetected (i.e., using a selective ion monitoring mode (SIM)), oralternatively, ions may be detected using selected reaction monitoring(SRM) or multiple reaction monitoring (MRM) (MRM and SRM are essentiallythe same experiment). Ions can also be detected by scanning the massanalyzers to detect all ions from the sample.

In one embodiment, the mass-to-charge ratio may be determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency (RF) fieldexperience a force proportional to the amplitude of the RF signal, thedirect current (DC) potential applied between electrodes, and the ion'sm/z ratio. The voltage and amplitude can be selected so that only ionshaving a particular m/z travel the length of the quadrupole, while allother ions are deflected. Thus, quadrupole instruments can act as a“mass filter,” a “mass separator” or an ion lens for the ions injectedinto the instrument.

One can often enhance the resolution of the MS technique by employing“tandem mass spectrometry” or “MS/MS” for example via use of a triplequadrupole mass spectrometer. In this technique, a first, or parent, orprecursor, ion generated from a molecule of interest can be filtered inan MS instrument, and these precursor ions subsequently fragmented toyield one or more second, or product, or fragment, ions that are thenanalyzed in a second MS procedure. By careful selection of precursorions, only ions from specific analytes are passed to the fragmentationchamber (e.g., a collision cell), where collision with atoms of an inertgas produce these product ions. Because both the precursor and productions are produced in a reproducible fashion under a given set ofionization/fragmentation conditions, the MS/MS technique can provide anextremely powerful analytical tool. For example, the combination of ionselection or filtration and subsequent fragmentation can be used toeliminate interfering substances, and can be particularly useful incomplex samples, such as biological samples.

In another embodiment, the mass-to-charge ratio may be determined usinga hybrid mass spectrometer system containing an electrostatic ion trapmass analyzer capable of high resolution and accurate massdetermination, for example a Q-Exactive™ mass spectrometer system(Thermo Fisher Scientific) which contains a quadrupole mass analyzer andan Orbitrap™ mass analyzer. Here, ions are selected by the quadrupolemass analyzer, then passed into a trapping device where the given ionpopulation is collected, collisionally cooled, and injected at highenergy and precise trajectory into the Orbitrap mass analyzer.Alternately, precursor ions are selected by the quadrupole massanalyzer, passed to a collision cell where product ions are produced,which are then passed into a trapping device where the given ionpopulation is collected, collisionally cooled, and injected at highenergy and precise trajectory into the Orbitrap mass analyzer. Ionsoscillate axially across the trap at a frequency proportional to(z/m)^(1/2) where z is the charge on the ion and m is the mass. Theimage current of these oscillating ions is detected and that frequencydomain data is converted into mass spectral information using theprinciple of Fourier transforms. The longer the transient collectiontime, the high the resolution for the subsequent mass spectral data.High resolution data can be obtained at values in excess of 200,000 withmass accuracies of 5 ppm or better.

For example, a flow of liquid solvent from a chromatographic column,possibly containing one or more analytes of interest, enters the heatednebulizer interface of a LC-MS/MS analyzer and the solvent/analytemixture is converted to vapor. Ions derived from the analytes ofinterest may be formed in the liquid phase and subsequently ejected intothe gas phase by nebulization in the ESI source or by reactions betweenneutral analytes and reactive ions as the analytes enter the gas phase.

The ions pass through the orifice of the instrument and passes a rangeof lenses, quadrupole, hexapole and similar devices prior to enteringthe instrument. In one embodiment, selected m/z windows of any m/z value(e.g., a 3, 5, 10, 20, 30, 40, 50, 100, 1800 or more dalton range ofm/z) may be analyzed to determine the molecular weights of the intactproteins in the window(s). In general, smaller m/z window sizes mayimprove signal-to-noise. In addition to the above stated m/z windowsizes, the m/z window size may be adjusted dynamically anywheredepending on experimental conditions. In another embodiment,pre-determined ion(s) from the window(s) are allowed to pass into thecollision cell where they collide with neutral gas molecules (e.g.,argon, nitrogen, or the like) and fragment. The fragment ions generatedare passed into the mass analyzer where the fragment ions are separatedand forwarded to the detector. In other embodiments, other fragmentationprocesses may include, but are not limited to, the absorption ofinfrared photons via infrared multiple photon dissociation (IRMPD), theabsorption of a single UV photon, through ion-ion reactions includingelectron transfer dissociation (ETD), or collisional-activation ofelectron transfer product ions which do not undergo promptfragmentation, electron capture dissociation (ECD). In an exemplaryembodiment, the dissociation method is the high energy collision-induceddissociation (HCD). As ions collide with the detector they produceanalog signal which is further converted to a digital signal.

The acquired data is relayed to a computer, which plots voltage versustime. The resulting mass chromatograms are similar to chromatogramsgenerated in traditional HPLC methods. Concentrations of the analytes ofinterest may be determined by calculating the area under the peaks inthe chromatogram, if there are any chromatographic peaks, or using theintensity of peaks in mass spectrum. The concentration of the analyte oranalytes of interest (e.g., proteins) in the sample is accomplished viaone of many different techniques know in the state of the art involvingexternal or internal calibrations, relative quantitation, peak height orarea counts, standard addition, or any other method known in the stateof the art.

A. Identification of Microorganism(s)

I. Microorganism Disruption and Solubilization of Proteins

Referring again to FIG. 1A, as shown in step a 102, a sample suspectedof containing one or more microorganisms may be disrupted and treated toobtain microbial cells directly from the sample (for example, urine), ormay be used to isolate pure cultures. Microbial cells are then used toproduce a soluble fraction of proteins. The sample may be of any typesuspected to contain one or more microorganisms including, withoutlimitation, isolated colonies from a culture plate; cells from liquidgrowth medium; blood, saliva, urine, stool, sputum, wound and body siteswabs; food and beverage; soil, water, air; environmental and industrialsurface swabs.

Cell disruption may be achieved by mechanical, chemical, enzymatic meansas are commonly known in the art and discussed in greater detail abovewith respect to FIG. 2. After disruption, the insoluble portion of thesample (typically cell wall material, certain lipids, precipitatedproteins, and other cellular components) may be removed from thesolution via centrifugation, filtration (either manual or automated) orother methods known in the art. Sample preparation may be automatedusing robotic systems controlled by one or more computers (see FIG. 2,202 and 215). Such robotic systems may be part of a larger system andlinked to other devices or computers.

In one example, a 1 mm bacteriological loop is used to collect cells ofactively growing E. coli from the surface of a suitable culture plate,for example OXOID™ Tryptone Soya agar plate (Thermo Fisher Scientific).The cells are suspended in 70% ethanol in LC/MS grade water and, aftertreatment for several minutes, the sample is centrifuged and supernatantis discarded. The cells are then subjected to lysis using 2.5%trifluoroacetic acid in ACN:water (1:1), after which the sample iscentrifuged for about 5 minutes at 14,000 rpm to remove insolublecomponents. Following centrifugation, the supernatant is transferred toa new tube and may be routinely fully evaporated either by using aspeedvac, or under a flow of nitrogen. Prior to analysis, the sample isreconstituted either in 2% ACN, 98% water with 0.2% formic acid (whenthe use of chromatography is anticipated), or in ACN:water (1:1) with 2%formic acid for flow injection or direct infusion. The reconstitutedsample is then subjected to direct analysis by high resolution massspectrometry or undergoes fast partial chromatographic separation andanalysis by high resolution mass spectrometry.

In another example, a 2 mm bacteriological loop may be used to collectapproximately 10 mg (wet weight) of cells of actively growing culture(e.g., Escherichia coli, Staphylococcus xylosus, Kocuria rosea, Bacillusliheniformis, Mycobacterium smegmatis or other) from the surface of asuitable culture plate, for example OXOID™ Tryptone Soya agar plate(Thermo Fisher Scientific). The cells are transferred to a 0.5 mlmicrocentrifuge tube and 20 μl of solution of 50% formic acid in 25%acetonitrile, 25% water is added; the pipette volume is increased to 40μl; and the suspension is vigorously pipetted up and down until thecells are disrupted, as indicated by the appearance of a foam. Then 180μl of acetonitrile:water (1:1) is added and the resulting solution iscentrifuged for approximately 5 minutes at 14,000 rpm. The supernatantis removed and diluted as needed for either direct infusion or flowinjection.

In another example, centrifuge is used to collect cells of Candidaalbicans from the liquid growth medium, for example Sabouraud liquidmedium (OXOID™, Thermo Fisher Scientific). The cells are washed from thegrowth medium with 0.9% physiological saline three times and sedimented.The cells may then be pre-treated with a mixture of ethanol and methyltert-butyl ether (7:3) at room temperature for 10 min or less. The cellsare sedimented by centrifugation and supernatant is discarded. A mixturethat includes about 70% formic acid, 15% acetonitrile and 15% water maybe used to lyse the cells and solubilize proteins. Insoluble componentsare sedimented at 14,000 rpm for 5 min, supernatant is transferred to aclean vial or centrifugal tube and is diluted with 0.2% formic acid inacetonitrile:water (2:98) prior to chromatography. When nochromatography is anticipated, the supernatant is diluted in a way toadjust concentrations of solvents to acetonitrile:water (1:1), 0.2-2%formic acid. Instead for acetonitrile, methanol can also be used. Aftercentrifugation, the diluted supernatant is then subjected to in-linesolid phase extraction with or without fast partial chromatographicseparation and analysis by high resolution mass spectrometry.Alternatively, the sample in acetonitrile:water (1:1), 0.2-2% formicacid is either flow injected, or directly infused into a massspectrometer for the analysis.

II. Sample Desalting, Concentration and Chromatographic Separation

The supernatant produced by disruption of the microorganisms in step 102contains intact proteins that may be further processed to desalt andconcentrate the proteins, as illustrated in step 104 of FIG. 1A. In oneembodiment, an automatic solid phase extraction/liquid chromatographysample introduction interface system is used to simultaneously desalt,concentrate and separate the intact proteins. A schematic diagram ofsuch a system 300 is shown in FIG. 3. In a first flow path 302, thesystem 300 may employ a single-use disposable solid phase extraction(SPE) cartridge 304 that is coupled to a pump 306, fluid lines 308, afirst switching valve 310, a second switching valve 312, and anelectrospray ionization (ESI) emitter 314. The SPE cartridge 304 may beconditioned, for example, using a 2% acetonitrile/0.2% formic acidaqueous solution (loading buffer). Next the sample prepared in step 102of FIG. 1A may be loaded from substantially the same solution and passedthrough the SPE cartridge 304 using, for example, reverse flow. The SPEcartridge 304 may then be washed with 2 bed volumes of the loadingbuffer to remove salts and other contaminants. After the washing step,each sample is eluted from the SPE cartridge 304 in a solvent volumewhich may be as small as 10 nl or less or as large as 10's of μl toconcentrate and optimize the intact proteins for delivery to the ESIemitter 314 and the mass spectrometer 316.

In one embodiment, the on-line SPE cartridge may be a polypropylene tipwith a small volume of silica sorbent containing bonded C₄, C₈, C₁₈ orother functional groups immobilized in the tip, for example, a Stage™tip (Thermo Fisher Scientific). In alternative embodiments, polymericsorbents or chelating agents may be used. The bed volume may beapproximately 1 μL to 1 ml. The apparatus and method are well suited tothe complex samples derived from the microbial cells because each SPEcartridge is used only once, minimizing carryover problems from onesample to another.

III. Ionization, Mass Spectrometry Analysis, Amino Acid SequenceInformation

As shown in step 106 of FIG. 1A, the sample is then subjected to massspectrometric analysis. The eluted proteins are ionized, for example,via electrospray ionization (ESI) or other atmospheric orsub-atmospheric pressure ionization techniques that can be readilycombined in-line with liquid chromatography. For ESI, proteinsaccumulate multiple charges based on the number of free N-termini,histidine, lysine, arginine, or other charge carrying amino acidspresent in the sequence. The resulting mass spectrum reflects thedistribution of multiply-charged ions (“charge state envelope”)originating from the same protein that appear at different mass/charge(m/z) values. The m/z values are the result of the same analyteacquiring different number of charges from the electrospray ionizationprocess. The distributions of the charge states are amenable fordetection via single or multiple stages of high resolution massspectrometry whether the sample comprises intact proteins and/or theirfragments produced via a top-down method. The molecular weight of aprotein can be calculated in a variety of ways. This includes simplemethods such as looking at spacing between adjacent isotopes of a singlecharge state, determining or calculating it using the m/z values ofseveral neighboring isotopically unresolved peaks derived from the sameprotein, and determining the distance in m/z space between those peaks.Other methods for calculating molecular weights include the thrashalgorithm and maximum entropy approaches as well.

After ionization the proteins are passed to a mass analyzer foranalysis. As shown in steps 108 and 110, the sample is repetitivelyscanned in full-scan high resolution MS mode within a selected m/zrange. In one embodiment, the mass spectrometer is repetitively scannedin the full-scan high resolution MS mode, for example, in a range fromm/z 150 to m/z 2000 in approximately one second to provide massmeasurement of the intact proteins at a mass accuracy of approximately 5parts-per-million (ppm), 3 ppm, 1 ppm, or better. In this embodiment,the source parameters are set as follows: spray voltage=4 kV, capillarytemperature=270° C. and source temperature=60° C. For the exemplary LCflow rate 400 μl/min, the source sheath gas is provided at the flow rateof 35 (arbitrary units) and auxiliary gas at the flow rate 5 (arbitraryunit), and source temperature=60 C.°. The obtained mass accuracy issufficient to provide elemental composition information for the protein(e.g., the number of carbon, nitrogen, oxygen, hydrogen, sulfur, orother atoms present in the protein). This information can be furtherused as part of an algorithm to identify microorganism.

As used herein, the terms “mass accuracy” and “ppm” refer to the degreeof conformity of a measured quantity to its actual true value. In thecase of measuring the mass, or more specifically the mass-to-chargeratio, of an ion in a mass spectrometric measurement, the difference orerror between the experimentally measured mass and the exact mass of anion is expressed in units of parts-per-million (ppm) according to theequation: ((measured mass−exact mass)/(exact mass))×10^6=mass accuracyin ppm.

The exact mass of an ion is obtained by summing the masses of theindividual isotopes of the molecule, including any correction for thecharge state of the ion. For example, the exact mass of an ionized watermolecule containing two hydrogen-1 atoms, one oxygen-16 atom andcarrying a charge of +1 is:((1.007825+1.007825+15.994915)−0.000549)=18.010016 Daltons. If themeasured mass of this ion was 18.010200 Daltons (Da), then the accuracyof that measurement would be: ((18.010200 Da−18.010016 Da)/(18.010016Da))×10^6=10.2 ppm.

Measurement of the mass of an unknown protein or any protein fragmentderived biologically or from MS^(n) to an accuracy of 5 ppm, or better,reduces the number of candidates found when searching a database ofknown proteins.

In FIG. 4 a full scan electrospray mass spectrum of an E. coli extractperformed via direct infusion is illustrated. The scan ranges from m/z400 to m/z 1800. The extract was obtained as described in thisapplication and was run without any further purification or desalting.The peaks present primarily represent multiply charged mid- and highmass proteins with the exception of some small molecules, small peptidesand lipids. This scan provides quality control information that theinstrument is functioning properly and the signal is stable for furtherdetailed analysis. Total time of analysis is one second.

Mass isolation of a 50 Da window indicated in FIG. 4 is shown in FIG. 5.The mass range scanned is from m/z 750 to m/z 800. Present in the figureare more than nine isotopically resolved peaks representing differentcharge states of nine different proteins at the following m/z values:759.4283 (+18), 761.7664 (+14), 766.8419 (+12), 769.7618 (+12), 771.5050(+10), 782.8381 (+8), 788.1067 (+10), 793.2172 (+13), and 795.5254(+12). These charge states are then converted to molecular weight valuesbased on the charge state specific isotope spacing. This yields thefollowing molecular weights: 13651.71, 10735.79, 9190.10, 9237.14,7705.05, 6254.70, 7871.07, 10311.82, and 9546.30. These molecularweights can be searched against the pathogen molecular weight databasein real time in order to retrieve a reduced number of potential pathogenidentifications. For example, 50S ribosomal protein L27, nucleoidassociated protein YbaB, 50S ribosomal protein L17, integration hostfactor subunit b, 30S ribosomal protein S16, uncharacterized proteinYehE, and r50S ribosomal protein L31 were tentatively identified fromthis search where the input data originate from high resolution massspectrometry that delivers very high measurement mass accuracy.

The identification process can occur in real time during the dataacquisition or post acquisition. The data acquisition process defined instep 110 of FIG. 1A can include the direct infusion of the proteinextract or flow injection combined to SPE clean-up as described in thisapplication. The key component here is matching calculated molecularweight obtained experimentally with those in the microorganism/pathogendatabase. When the identification process occurs during the dataacquisition period, the highly accurate molecular weight informationobtained for a defined acquisition window is matched against the highlyaccurate molecular weight microorganism/pathogen database. Thiseffectively reduces the number of possible microbial identificationscandidates in real time (in several seconds).

In another embodiment of the invention, proteins are analyzed not onlyby their molecular weight, as described above, but also using the dataon their unique amino acid sequences which are then compared to areference database that contains known amino acid sequences of microbialorigin. In this embodiment, molecular weights of intact proteins aredetermined, and protein sequence information is obtained via multi-stagemass spectrometry. This is illustrated in step 112 of FIG. 1A. Using themolecular weights determined in steps 108 and 110 allows for the followon step of tandem mass spectrometry to unequivocally identify themicroorganism/pathogen of interest. Molecular weights can be calculateddirectly from distances between the isotopes in isotopically resolvedpeaks, or by determining the centroid and m/z spacing for very high massproteins (peaks are not isotopically resolved) as the data acquisitionprogresses.

Typically, a selected charge state of a protein is mass isolated andexcess energy is deposited to a protein precursor ion population inorder to induce the formation of sequence specific fragment ions as aresult of collisions with an inert gas (atomic or molecular) or by othermethods known in the art. This energy deposition process may be derivedfrom low or high energy collisional activation (CA) dissociation event,the absorption of infrared photons via infrared multiple photondissociation (IRMPD), the absorption of a single UV photon, throughion-ion reactions including electron transfer dissociation (ETD) orcollisional-activation of electron transfer product ions which do notundergo prompt fragmentation, electron capture dissociation (ECD), orother.

In an exemplary embodiment, the dissociation method is low or highenergy collision-induced dissociation (CID) where normalized collisionenergy ranges from 5 to as high as 50 percent (normalized collisionenergy, arbitrary units). Precursor ions are typically automaticallyselected from the most intense peak in a charge state distributionderived from any given protein.

FIG. 6 illustrates a tandem mass spectrometry scan of the of the 50 Dawindow from m/z 750 to m/z 800 described above with respect to FIGS. 4and 5. All precursor ions listed in FIG. 5 were subjected tocollision-induced dissociation (CID) via the HCD cell on a Q Exactivehigh resolution/mass accuracy mass spectrometer. The normalizedcollision energy was set to 18% with a resolution of 35,000. Theresulting fragment ions produced ranged in charge state from +1 to +9for the thirty most prominent peaks. These peaks correspond to fragmentions derived from a representative set of labeled (via charge state)precursor ions shown in FIG. 5. These fragment ion identities are thenmatched to the precursors listed above but can be associated withprecursor ions with very low signal-to-noise ratios. This matchingprocess also occurs in real time and is used to narrow theidentification of the pathogen to E. coli.

However, in one embodiment of the invention the more highly chargedstate ions of a given protein are chosen to undergo CID. This issupported by the data shown in FIG. 7. Here the resulting product ionsare obtained as a resolution of 35,000 with a mass accuracy of 3 ppm orbetter. The protein shown was identified as DNA Binding Protein H-nsfrom a resistant E. coli strain grown in the presence of ampicillin. Themethod of collision-induced dissociation (CID) was used to fragment theintact protein at a precursor m/z value of 811.9018 (+19 charge state,approximate molecular weight 15.4 kDa). By selecting this precursor ion,the resulting fragments (b-y series ions) derived from the intactprotein can be identified via preferential cleavage sites. In thisexample, the m/z values 813.82440, 904.13885, and 1017.03210 are b₇₀ions that cleave between an aspartic acid (D) and proline residue (P).Other prominent peaks at m/z 742.90741 and 1077.57056 cleave on theC-terminal side of glutamic acid (E) to produce b₂₆ and b₂₇ ions. Thefragment matching portion of the algorithm preferentially weights moreintense fragment ions to accurately identify the protein quickly. Bycombining this information with the molecular weight of the intactprotein, protein can be identified and pathogens can be identified at tothe species level and, in many cases the strain level.

In one embodiment, sequence tag information may also be generated toconfirm the identification of the protein. A computer software programexamines the product ion mass spectrum for obvious sequence tags. Asequence tag is a short string of two or more amino acids deduced bymass differences between major fragment ions in the product ion massspectrum. The sequence tag and its location in the peptide or proteinrelative to the amino and carboxyl terminus are used as constraints inthe database search. Used in combination with molecular weight, sequencetag information provides for identification of proteins with highconfidence. Since many proteins produced in prokaryotic and eukaryoticcells undergo loss of N-terminal methionine, signal peptides, or otherpost-translational modification events, calculations are used to accountfor these modifications. This principle is also illustrated in FIG. 7.FIG. 7 illustrates a series of singly-charged y-type ions whichcorrespond to the sequence tag I/L I/L F D. This in combination with themolecular weight at 15.4 kDA can be used to identify the protein. Inaddition, the most intense fragments observed are those where cleavageoccurs on the C-terminal side of aspartic (D) and glutamic acid (E) andthe N-terminal side of proline (P). These preferential cleavagestypically observed in the CID mass spectrum of proteins can be used tospeed up the database search process by weighting these peaks based onintensity.

Additionally, programs such as ProSight PTM, MS-Align, UStag,MS-TopDown, PIITA, and OMMSA (Open Mass Spectrometry Search Engine) canbe used to identify intact proteins derived from top-down massspectrometry experiments. Smaller peptide and protein fragments uponmass analysis of the corresponding product ions are identified via oneof several different database search engines including correlation based(Sequest), probability based (Mascot), expectation value calculationprograms (!Xtandem), or other approaches know in the art. Proteinidentifications from the aforementioned methods are then used toidentify the genus, species, subspecies, strain and/or serovar of theorganism. The same protein identification workflow method can be appliedto proteins specific to virulence factors, resistance markers, and otherrelevant markers.

In another embodiment of the invention, tandem mass spectrometry is usedto generate fragment ions of the peptide or protein, and the resultingspectrum is matched against a multi-stage mass spectrometry referencedatabase to identify the organism. Such database may also includechromatographic retention time information, mass of the proteinincluding any posttranslational modifications, mass of the peptide orprotein fragments, elemental composition (C, H, N, O, S or other atoms),general peak intensity or intensity as it relates to preferentialcleavage, and sequence tag information derived from knownmicroorganisms.

IV. Data Analysis and Identification to Genus-Species Level

Any one or more of chromatographic retention time, mass, intensity,elemental composition, amino acid composition, and protein sequenceinformation may be used to identify the microorganism(s) present in thesample. Identification is based on MS and MS^(n) data and any of theaforementioned parameters compared to a known reference database(s).Chromatographic retention time may be absolute, as measured using adefined column and set of chromatographic conditions to separate thecomponents or group of the components of the sample, or may be relativeto the retention time of some other component or components present inor added to the sample being analyzed.

The steps of an algorithm for providing an initial ID for an unknownmicroorganism in a sample may be performed in less than a minute. Theidentification process can occur in real time during the chromatographicseparation and data acquisition or after data acquisition and/orchromatographic separation. During the data acquisition periodparameters such as retention time, mass-to-charge ratio (m/z), andintensity information is stored in the systems on-board memory. When theidentification process occurs during the chromatographic separation/dataacquisition step, raw data is processed in real time based on theprinciples of MS/MS (tandem mass spectrometry) as well as molecularweight matching by an identification algorithm. An identificationalgorithm involves matching the experimental peaks based on retentiontime, mass, elemental composition, amino acid frequency, and intensityto a reference database. Once enough data has been acquired to identifythe microorganism(s) of interest, confirmation of identification and/ordetection of virulence factors and resistance markers may be performed.For the MS/MS process, confirmation peaks are chosen in real time basedon the identification of the microorganism. The MS/MS process occursautomatically and the results are searched against a MS/MS database,which contains sequence information for known microorganisms.

Alternatively, peaks can be directly identified using another algorithm.The same principle applies to the confirmation of virulence factors orresistance markers for a particular organism. Once all data is acquired,MS-based information and MS/MS-based protein/microorganismidentification information are updated in real time and are used in ascoring process performed using an algorithm specifically developed forsuch purpose.

Referring now to FIG. 1B, one example of a suitable algorithm 130 thatcan be used to identify a microorganism based on the data obtained insteps 102 through 112 (see FIG. 1A) and steps 110 and 112 (See FIG. 1A)in particular is illustrated. The algorithm includes a step 132 ofobtaining mass-spec data for molecular weight determination. This may bedone in step 110 of FIG. 1A. The algorithm then includes a step 134 ofpeak detection, a step 136 of determining whether any of the peaks areisotopically resolved. If the peaks are isotopically resolved, thealgorithm calculates the molecular weight associated with the peaks instep 142. If the peaks are not isotopically resolved, the algorithm thenincludes a step of checking for the presence of other unresolved peaksthat can be assigned to the same charge state envelope. If there areadditional peaks, molecular weight can be calculated according to step142. If there are no additional peaks in the same mass spectrum, thealgorithm looks for the candidates in the mass spectra that show theadjacent mass ranges. If the peaks are not found, the original peaks areremoved from the analysis in step 140.

Based on the calculated molecular weights (step 142), the algorithm 130may then create a molecular weight search list (step 144) and search themolecular weight search list against a molecular weight/microorganismdatabase (step 146). The algorithm 130 then asks whether or not all ofthe molecular weights match in step 148. If the molecular weights do notmatch, the algorithm 130 creates an unknown list for post MS/MS matching(step 150) and creates a target identification list (step 152). If themolecular weights do match, the algorithm creates a targetidentification list (step 152).

The algorithm 130 is capable of using the information derived about themolecular weights of the protein in the sample derived in the previousto perform subsequent analysis to refine the assignment of the identityof the organism. In step 154, the algorithm may direct the mass-specinstrument to perform a tandem mass-spec on the peaks acquired in step132. The algorithm then directs the creation of a tandem MS fragmentdatabase from the target identification list of step 152 in step 156,and then in step 158 match the fragments from step 154 against thedatabase from step 156. These data (steps 152 and 158) are used toidentify the microorganism in step 160.

The algorithm 130 then checks whether or not this is a match to a singleID in step 162. If there is a single match in step 162, the algorithm130 then includes an optional step of matching masses from the listgenerated in step 150 against possible post-translational modificationsand/or errors in the database annotation. The sample may be submittedfor further analysis as described herein below as illustrated in step172. If the match in step 162 is not to a single ID, the algorithm thenrepeats steps 132 through 160 with a different mass range used in step132. If the match is the same as obtained in step 162, then the samplemay be submitted for further analysis as described herein below asillustrated in step 172. If the match is not the same as obtained instep 162, then the sample may be flagged for further analysis (step168).

All processes of identification may be fully automated and occur in thebackground of a given data acquisition process. Alternatively, thesesteps can occur post acquisition as well. The results of microorganismidentification may be provided in a user friendly format, for example,to a remote device, a mobile device and/or a centralized computersystem, which can be pre-selected prior to sample processing.

Alternative approaches may be used to match high resolution/massaccuracy ESI data to the spectra in reference databases. Such methodsinclude, without limitation, linear, randomized, and neural networkpattern match based approaches. Electrospray ionization allows a greaternumber of proteins from any given organism to be detected, so that thespecificity of the pattern match based approach is greatly improved overMALDI-based techniques. The increased information content available withthe LC-MS and MS/MS based approach of the present invention allows for amore detailed characterization of the microorganism at the strain orsubspecies level and can minimize the false positive rate. For example,the presence of two or more different species, strains, or subspecies ina single sample has presented a challenge to previous analyses basedupon MALDI-TOF mass spectrometry. An advantage of the combined MS andMS/MS (MS^(n)) approach of the present invention is that any uniqueprotein may be used to check the accuracy of identification. A databasemay include information regarding species capable of unequivocalidentification based upon presence of a single protein. Speciesrequiring further checks can be analyzed independently.

B. Targeted Analysis of Specific Characteristics

When identification at the genus/species level is accomplished, thesample may optionally be reanalyzed for detailed information including,without limitation, information relevant to typing (strain and/orserovar), virulence, or antibiotic resistance and/or susceptibility.This second analysis (reanalysis) may be targeted for one or morespecific proteins based upon the identity or identities of themicroorganism(s) identified during the first analysis. This is indicatedin FIG. 1A by arrow 114, which shows information from the first analysisbeing used to direct the second analysis.

Based upon the genus or species identification obtained from thesequence of events in steps 104 through 112 of FIG. 1A, the instrumentsoftware may use programs coded with the Instrument Advanced ProgrammingInterface (IAPI). The IAPI software (Thermo Fisher Scientific) is a dataacquisition directing master control program that allows the programmingof the logics of making decision for the next acquisition steps inreal-time while the sample is still being analyzed and the analyte isstill readily accessible for further analysis. The software is .NETcompatible event driven software that virtually adds no overhead time tothe analysis. Since the software system has asynchronous control,multiple “listeners” can respond to s single event or trigger. Anyvariety of computer language can be used including all derivative of theC programming language, visual basic, and Python just to name a few. Asan example, the software is used to drive the molecular weight databasereduction process that is described earlier and is performed during dataacquisition. In this section is described the methodology for triggeringa selective experiment for typing, virulence detection, and resistancemarker identification.

As an example for the case of pathogenic E. coli, the software canimmediately set up a rapid scan for detection of the appropriateexpressed virulence, resistance, or typing markers. For E. coli thiswould include adhesions, invasions, motility/chemotaxis, toxins,antiphagocytic proteins/molecules, and proteins involved in suppressingthe immune response. These can be monitored using fast partialseparation mass spectrometry (FPCS-MS) and targeted tandem massspectrometry as described in the following section of this application.

I. Solid Phase Extraction

Referring to step 116 of FIG. 1A, a second injection of soluble proteinsextracted from a sample suspected of containing one or moremicroorganisms (either the same extract used for identification purposesas described above or another extract prepared from the same sample) maybe subjected to a second solid phase extraction (SPE) procedure.

Referring again to FIG. 3, for the second SPE procedure, a system 300may include a second flow path 318 that can be used to couple an SPEcartridge 320 to a chromatography column 324 via a fluid conduit 322, afirst switching valve 326, a second switching valve 328, and anelectrospray ionization (ESI) emitter 314. The SPE cartridge 320 wasdescribed above in connection with step 104 of FIG. 1A. The SPEcartridge 320 is first conditioned, for example, using 100% methanol oracetonitrile or other suitable combination of solvents followed by a 2%acetonitrile/0.2% formic acid aqueous solution (loading buffer). Nextthe sample is loaded from a substantially identical solution is loadedand passed through the SPE cartridge 320 using reverse flow. The SPEcartridge 320 may then be washed with 2 or more bed volumes of theloading or other LC/MS compatible buffer to remove salts and othercontaminants.

After the washing step, the sample may be eluted from the SPE cartridge320 in a solvent volume which may be as small as 10 nL or less or 10'sof μL to concentrate and optimize intact proteins for delivery to thechromatography column. The SPE cartridge is then placed in fluidconnection with the chromatography column 324 for fast partialchromatographic separation of the intact proteins derived from themicrobial cells.

II. Fast Partial Chromatographic Separation Mass Spectrometry (FPCS-MS)

Referring to step 118 of FIG. 1A, the sample is then subjected topartial chromatographic separation followed with mass spectrometricanalyses. Generally, in performing FPCS-MS, a crude extract of microbialcells containing a complex mixture of various organic and inorganicanalytes (small organic molecules, proteins and their naturallyoccurring fragments, lipids, nucleic acids, polysaccharides,lipoproteins, etc.) is loaded on a chromatographic column and subjectedto a chromatography. However, instead of allowing a gradient to eluteeach analyte separately (ideally, one analyte per chromatographic peak),the gradient is intentionally accelerated to the extent thatsubstantially no chromatographic peaks obtained for exampleapproximately 8 minutes or less, and preferably 5 minutes or lessinstead of a much longer run time that would be required to obtain abaseline separation. Instead, many analytes are intentionally co-elutedfrom the column at any given time according to their properties and thetype of chromatography (reverse phase, HILIC, etc.) used. Partial orincomplete separation may be also accomplished by other methods known toone skilled in the art, including but not limited to the use of mobilephase solvents and/or modifiers that reduce retention of compounds onthe column, selection of stationary phase media that reduce retention ofcompounds on the column (including particle size, pore size, etc.),operation of the chromatographic system at higher flow rate, operationof the chromatographic system at an elevated temperature, or selectionof a different chromatographic separation mode (i.e., reversed-phase,size exclusion, etc.).

Since there are substantially no chromatographic peaks across the wholegradient substantially all of the information about the analytes in amixture is obtained from the mass spectra. Substantially the onlyrelevant information derived from a chromatogram is the time of elutionfrom the column. Each mass spectrum that is recorded represents a“subset” of co-eluting analytes that is then ionized, separated in massanalyzer and detected. Because all co-eluting analytes are ionized atthe same time, they are the subject to the known effects of ionizationsuppression in mixtures that result from but are not limited to: (1)competition for the charge and (2) suppression of the signal from lessabundant analytes. Although effects of ionization suppression aregenerally referred as “undesirable” for many mass spectrometry methods,in FPCS-MS these effects are used for the benefit of the analysis.However, due to the effects of ionization suppression/suppression of“the less abundant” by “more abundant,” the signal from a significantlyreduced number of proteins is recorded in a mass spectrum.

FIGS. 11A-11C illustrate FPCS-MS total ion current profiles of variousmicroorganisms grown on the Oxoid Tryptic Soy Agar at 34 C for 20 h.(A)—Escherichia coli ATCC 8739; (B)—Enterococcus gallinarum ATCC 700425;(C)—Bacillus subtilis subs. spizizenii ATCC 6633. The total informationabout the analytes in a sample is compiled from the data which areextracted from each mass spectrum across the whole chromatographic runthus representing all co-eluting “sets” of analytes. As there arepractically no chromatographic peaks (for example, 3-5 broad peaksacross the whole 5 min gradient, as it is illustrated in FIGS. 11A-11C),all necessary information is derived from the mass spectra (m/z,intensity) with the only information that originate from the LC run, isthe time of co-elution of the “sets” of analytes (only some of theoriginal mixture components are represented in the mass spectrum due tothe ionization/separation effects, as described above).

FPCS-MS requires no special columns, or small columns, or unusuallyshaped columns (for example, V-shaped columns) in combination to highflow rates, as is generally practiced for “ballistic chromatography” or“ballistic gradients”. The columns used in FPCS-MS may be standardchromatographic columns. For example, the length of a column can be 20mm, 30 mm, 50 mm, 100 mm, 150 mm, 250 mm and so on and/or the internaldiameter of such a column can be 2.1 mm, 1 mm, 500 um, 150 um, 75 um andso on. Particle sizes and pore sizes are also standard as known in theart.

The flow rates that are used in FPCS-MS, are standard for the type of acolumn in use. For example, flow rate may be 900 μl/min, 400 μl/min, 100μl/min, 30 μl/min, 200 nl/min, and so on.

By varying the chromatographic conditions (column dimensions andchemistry, particle and pore size, mobile phases and modifiers of themobile phases), types of chromatography and off-line fractionation ofcomplex samples with the individual fractions still remaining complexmixtures, one can focus on very different sets of components of theoriginal complex mixtures.

As mass spectrum for the co-eluting analytes may be recorded from aslittle as just one mass spectrometer scan, the information which can beobtained about the analytes in mixture, is very abundant, as illustratedin FIG. 12.

In combination, the extremely high resolution and mass accuracy that isdelivered by the use of electrostatic ion trap which is the part of suchmass spectrometers as Q-Exactive, Exactive mass spectrometers (ThermoFisher Scientific) or similar mass spectrometers, provide a powerfultool for the discovery and targeted analysis of, for example, virulencefactors, biomarkers for antibiotic resistance and/or susceptibility,differentiation of strains and so on.

Overall, FPCS-MS provides rapid analysis, maximizing the number ofsamples that can be analyzed in a set period of time, while providingthe necessary information about the sample.

In a preferred embodiment, the mobile phase composition in areversed-phase chromatographic separation is ramped in a much more rapidfashion for the chromatographic column used resulting in proteins ofwidely varying molecular weights eluting from the chromatographic columntogether.

The stationary phase in the chromatography column may be porous ornon-porous silica or agarose particles, or a monolithic materialpolymerized or otherwise formed inside the column. The stationary phasemay be coated with an appropriate material such as C₁₈, C₈, C₄ oranother suitable derivative to facilitate the separation of theproteins, and such material may be chemically bonded to the particles ormonolith inside the column. Particle sizes typically range from about1.5 to 30 μm. Pore sizes can range from 50 to 300 angstroms. Insidediameters of columns typically range from about 50 μm to 2.1 mm, andcolumn length from about 0.5 cm to 15 cm and above. The mobile phase oreluent may be a pure solvent, or a mixture of two or more solvents, andmay contain added salts, acids and/or other chemical modifiers. Theproteins are separated on the column or two sequentially or in parallel(as it is in the two dimensional chromatography) connected columns basedon one or more physiochemical properties, including size, net charge,hydrophobicity, affinity, or other physiochemical properties.Chromatographic separation methods include one or more of ion exchange,size exclusion, hydrophobic interaction, affinity, normal-phase,reverse-phase or other chromatography.

In one embodiment, a reversed-phase chromatographic separation isperformed on a 50-mm×2.1-mm internal diameter (ID) chromatographiccolumn packed with 1.9 um particles and pore size 175 angstrom (C₁₈stationary phase) using the following two mobile phases: 0.2% formicacid in water (mobile phase A) and 0.2% formic acid in acetonitrile(mobile phase B) at the flow rate 400 μl/min. Separation is performed ina 2-80% gradient of mobile phase B in mobile phase A within 2, 5 or 8min.

In order to accommodate rapid analysis and sample turnaround time, thegradient is run on the order of approximately 8 minutes or less in oneembodiment of the present invention. This compressed gradient formatresults in proteins with widely varying molecular weights elutingclosely together. Using this embodiment, proteins up to 70 kDa mass canbe detected. A significant advantage of this method is the improvedspecificity of detection obtained from higher mass ranges. The massrange of the ESI/MS-based method significantly exceeds that of MALDImethods, which have a practical upper limit of about 12-15 kDa mass,and, in addition, all of the proteins typically observed in a MALDIspectrum are found in the ESI/MS mass spectrum.

In the other embodiments, a chromatographic column with 0.32 mm ID orsmaller and packed with a C₄ stationary phase is used with a 20-60%gradient of mobile phase B (acetonitrile with 0.2% formic acid) inmobile phase A (water with 0.2% formic acid) at a flow rate ofapproximately 10 μL/min. The gradient elution time for thechromatographic separation may range from approximately 10 minutes to 20minutes, followed by a short re-equilibration time that is typicallyless than the separation time.

III. Ionization and Mass Spectrometry

Referring to step 120 of FIG. 1A, the sample may be ionized andsubjected to mass spectrometric analysis as described in greater detailelsewhere herein. Referring to FIGS. 8A-8E, protein extracts from B.Licheniformis, C. albicans, K. rosea, S. xylosus, and M. smegmatis werepartially separated by the FPCS-MS procedure described above. Theseparation was performed on a 5 cm×2.1 mm i.d. column packed withHypersil Gold C₁₈-like column with 1.9μ particle size and a porediameter of 170 angstroms. Solvent A was composed of 100% H₂O and 0.2%formic acid and solvent B was made up of 100% ACN and 0.2% formic acid.Starting conditions were 98% A and 2% B at a flow rate of 400 μL/min anda column temperature of 40° C. Tandem mass spectrometry was performedusing data dependent analysis of the top three most intense peaksassociated with the full scan analysis. Masses that underwent MS/MS wereplaced on the dynamic exclusion list for a period of 20 seconds. Theresulting tandem mass spectra were searched via version 3.0 of ProSightPTM software.

The data shown in FIGS. 8A-8E illustrate that the FPCS-MS works for awide range of organisms across different microbial genera. In addition,one will appreciate that the FPCS-MS procedure described herein allowsthe second analysis procedure of steps 116-122 of FIG. 1A to beperformed much more rapidly as compared to procedures where specificgradients and, in some cases, specific mobile phases are run fordifferent organisms. Likewise, because the procedure described hereindoes not depend on baseline chromatographic separation of the componentsof the mixture, the gradient can be run much faster (e.g., 5 minutes vs.30 minutes or vs. 90 minutes). It is actually a virtue of the methoddescribed that there is a lack of baseline separation and that thecomponents crowd together when they come off the column. For example, asdescribed above, the forced co-elution of subsets of proteins causessuppression of ionization of the mixture. Although ion suppression isgenerally considered to be disadvantageous, in this case ion suppressionhas the effect of simplifying the ion spectrum of the complex mixturecoming off of the column by virtue of the fact that a signal resultsfrom only the most ionizable and plentiful proteins in the recorded inthe mass spectrum.

IV. Strain Typing

As shown in step 122 of FIG. 1A, protein(s) specific to a given strainor serovar type may be used for typing individual isolates. Variationwithin microorganism isolates is often the result of deletion and/orinsertion of entire gene(s). Therefore, it follows that differentstrains may potentially lack or gain hundreds of strain-specificproteins and this variation may provide a uniquely discriminatory typingsystem.

In one example, twelve strains of E. coli were analyzed using themethods of the present invention. The ability of the present inventionto detect a bigger number of proteins, proteins of higher molecularweights and with better mass accuracy than those detected usingconventional MALDI methods was tested in connection with variations of a35.4 kDa protein found in each of the twelve strains. As shown in Table1, five different forms of a protein having a mass of approx. 35 KDawere detected.

TABLE 1 ATCC Strain of E. coli Mass of the common protein (Da) 1177535218 4157 14948 8739 33876 43888 51446 10536 11229 35421 29194 35,167 ✓✓ — — — — — — — — — — 35,176 — — ✓ ✓ ✓ ✓ ✓ ✓ — — — — 35,413 — — — — — —— — ✓ — — — 35,426 — — — — — — — — — ✓ — — 35,497 — — — — — — — — — — ✓✓

One can see from the Table 1, that the strains of E. coli can be groupedaccording to the common protein which yet has a different mass due tothe variations in the amino acid sequence. This protein has MW 35413 Dain ATCC 10536 and MW of 35426 Da in ATCC 11229 MW of 35497 Da wasobserved for this protein in two strains (ATCC 35421 and ATCC 29194).Likewise, this protein has MW of 35167 Da when isolated from two otherstrains, namely ATCC 11775 and ATCC 35218 and MW of 35176 Da was foundin five different strains. The protein was detected at the sameretention time in twelve ATCC strains of E. coli to illustrate theutility of the present invention for distinguishing between closelyrelated strains of microorganisms according to one embodiment of theinvention. The mass spectra contain also other proteins that vary indifferent strains (either the same proteins which amino acid sequencevary, or different proteins in different strains). Detection of multipleproteins as the part of identification procedure and use of tandem massspectrometry provides accurate results and the confidence when typingthe strains.

V. Characterization of Virulence Factors and Resistance Mechanisms

A second analysis may also be used to identify virulence factors orantibiotic resistance and/or susceptibility markers present in theidentified microorganisms. Such analysis may or may not requirepre-treatment of the sample, for example, a brief exposure to one ormore antibiotics or other stressful conditions (e.g., temperature,scarcity of one or more nutrients, iron deficiency, copper exposure,etc.) to induce proteins associated with resistance, susceptibility orvirulence. For analysis of virulence factors and/or resistance markers,the mass spectrometer is run in the target MS/MS mode (product ionscanning) to detect known virulence factors or resistance markers.

This principle is illustrated in FIGS. 9A-9D. In FIGS. 9A-9D, anantibiotic resistant strain of E. coli (ATCC 35218) was grown in thepresence and absence of antibiotics at 37° C. for 18 hours on the OxoidMueller-Hinton Agar (Thermo Fisher Scientific). In FIG. 9A, cells weregrown in the presence of oxacillin; in FIG. 9B, cells were grown in thepresence of naficillin; in FIG. 9C, cells were grown in the presence ofpenicillin; and in FIG. 9D, cells were grown in the presence ofampicillin. In each of the examples, the boxed regions indicate portionsof the chromatograms where significant changes are observed in massspectra obtained for the cells that originate from antibiotic treatedand not treated cultures. In the mass spec experiment, changes inprotein expression are shown. However, changes resulting from antibioticexposure may include alterations in protein expression, lipids, andsmall molecules either individually or combined to indicate antibioticresistance.

It is possible to detect certain resistance, sensitivity or virulencemarkers after a brief incubation with antibiotic of approximately 10minutes or less. Further, analysis of resistance markers and/orvirulence factors may be quantitative. The marker information is usefulfor patho-typing and characterization of a microorganism for purposes ofpatient treatment or for collecting epidemiologic information.

In one embodiment of the invention, top-down proteomics may be used toinvestigate the various forms of the resistance marker, β-lactamase.Multiple β-lactamase types (AmpC, ESBL, KPC, etc.) can occur in the samecell leading to errors in screening and confirmatory phenotypic tests.Resistance marker information may be used to verify and correctconventional phenotypic susceptibility results. For example, changes inporins and/or AmpC over expression can mimic the phenotype of ESBL. Thepresence of a KPC beta lactamase masks the phenotype of an ESBL. Theseproteins can be identified using the top-down method of the presentinvention since MS/MS methods are capable of detecting any change orsubstitution in the amino acid sequence of the intact protein.

A direct comparison to a traditional bottom-up procedure commonly usedby proteomics laboratories further emphasizes the advantages of thetop-down method of the present invention. By way of example, if theentire DNA sequence of the organism is known before analysis isperformed, one can judge whether the β-lactamase enzyme might beexpressed. If the β-lactamase is indeed expressed, then there is achance that one or more peptides associated with the enzyme can beidentified in a bottom-up proteomics experiment. If the peptide(s)identified is/are specific to that one β-lactamase in 1000 knownvariants of β-lactamase, then one can say unequivocally that a specifictype is confirmed. However, the detected and identified peptide mayhappen to be common to many different variants of β-lactamase. Evendistinguishing two E. coli strains wherein β-lactamase variants differby a few as two amino acids is limited, if not impossible, using abottom-up approach.

In another embodiment of the invention, unique forms of resistancemarkers, such as β-lactamases, may be used to facilitate identificationof a given microorganism. For example, a direct comparison of specifiedextended spectrum beta-lactamases (ESBL) across all known microorganismsreveals only two species with 100% homology, namely Pseudomonasaeruginosa and Acinetobacter baumannii. If the particular ESBL isexpressed and detected, then microbial identification is narrowed to twoorganisms without even considering any other proteins. Theidentification of one or more proteins may be sufficient to confirmidentification of each.

As a further example, a search of a common variant β-lactamase specificto E. coli against all known sequences for similarity may be performed.However, a slight modification of the enzyme yields a variant common tomany different microorganisms. The β-lactamase specific to E. coli maybe used to differentiate E. coli from Shigella. It is of note that the(β-lactamase of Shigella flexneri and Shigella dysenteriae contain aβ-lactamase comprising 286 amino acids which is the number found in E.coli β-lactamase; however the sequence of the Shigella version ofβ-lactamase differs by one amino acid (substitution of a glycine forserine) which is sufficient to provide for accurate identification ofthe microorganisms.

For samples comprising a mixture of microorganisms, the diagnosticinformation provided by a resistance marker sequence may be used todetermine the microorganism expressing each antibiotic resistancemarker. Other resistance markers capable of providing useful informationinclude, without limitation, DNA gyrases, aminoglycisidases, effluxpumps (SrpA and MFP), proteins involved in folate metabolism, and rRNAbinding proteins.

In another embodiment of the invention, specific marker panels of targetproteins may be used to determine the identity of a microorganism. Thesepanels may require monitoring the intensity profiles of specificproteins as a function of time. A hybrid tandem mass spectrometer thatemploys a single stage quadrupole followed by a series of ion transferdevices, a low energy collision cell, a C-trap, an HCD cell and orbitrapmass analyzer is used to identify target proteins. Proteinidentification is accomplished via MS/MS analysis of intact proteins andsearched against a reduced microbial proteome database containing onlyproteins of relevance to the specific microorganism or group ofmicroorganisms.

This principle is demonstrated in FIG. 10. Part of characterizing anidentified pathogen is to examine the potential of resistance markers,virulence factors, strain typing, and antibiotic susceptibility onpatient outcomes. In addition to identifying targets via full scan orselected ion monitoring, quantitation may be required in some instancesto determine if the levels of a virulence factor (for example adhesions,phospholipases, and secreted aspartyl proteases) are critical enough toaffect patient outcomes. Here in FIG. 10, high resolution/mass accuracydata of extracted ion profiles from four different proteins derived fromC. albicans is illustrated. These ions at m/z values of 539.68341 (+12),698.09351 (+6), 698.99127 (+10), and 703.70038 (+20) correspond toproteins with masses of 6.46, 7.27, 6.97, and 14.0 kDa spread across aretention time range between 3.5 and 8.0 minutes as shown in the inset.Quantitation can be accomplished using external or internal standardmethods, standards addition, or relative quantitative approaches.Examples include the use of label free techniques, selected reactionmonitoring, in-line spectroscopic approaches, metabolic labeling, orchemical labeling. Peak areas or heights can be used for amountcalculations along with values obtained for each charge state of a givenprotein by using resolved or unresolved isotopic clusters.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. A method for identifying one or more unknownmicrobes, comprising: a) providing a sample containing one or moreunknown microbes; b) disrupting the one or more unknown microbes presentin the sample to provide a fluid extract; c) separating a solubleprotein fraction from an insoluble protein fraction present in the fluidextract and preparing a solution containing soluble, intact proteinsfrom the one or more unknown microbes; d) injecting the solution into anion source of a mass spectrometer system and ionizing a stream or flowof the solution to form ionized proteins; e) analyzing the ionizedproteins with a mass analyzer of the mass spectrometer system, whereinanalyzing comprises: i) in a first mass spectrometry step, acquiring oneor more mass spectra representative of one or more of the solubleproteins in the sample; ii) determining molecular weights for theproteins from the one or more mass spectra; iii) using the determinedmolecular weights to search a first database containing molecularweights of known microbial proteins to identify, for a second massspectrometry step, a set of intact target proteins representative of asubset of candidate microbes; iv) in the second mass spectrometry step,selecting one or more precursor ions from the set of intact targetproteins and fragmenting the precursor ions in the mass spectrometer byfragmentation means to produce a plurality of product ions; v) usingmass-to-charge ratios of the plurality of product ions to search asecond database containing molecular weights of known microbial proteinsand at least one of product ion m/z values, amino acid sequence, orpost-translational modification information of said known microbialproteins, wherein the subset of candidate microbes from the firstdatabase is used to limit a search of the second database; and f) usinginformation obtained in step (e) for one or more proteins from each ofthe one or more unknown microbes in the sample to identify at least oneof the unknown microbes, wherein steps (b)-(f) are completed in lessthan 25 minutes.
 2. The method of claim 1, wherein step (b) is effectivefor Gram-positive bacteria, Gram-negative bacteria, yeast, viruses,mycobacteria and filamentous fungi.
 3. The method of claim 1 or claim 2,wherein the solution of soluble proteins is streamed directly into anionization apparatus.
 4. The method of claim 1, wherein the ionizationis electrospray ionization.
 5. The method of claim 1, wherein in thefirst mass spectrometry step the mass spectra for the one or more of theproteins in the sample have a mass accuracy of at least 15parts-per-million.
 6. The method of claim 1, wherein the fragmentationmeans is collision-induced dissociation.
 7. The method of claim 1,wherein in the second mass spectrometry step the mass-to-charge ratiosfor the product ions have a mass accuracy of at least 15parts-per-million or better.
 8. The method of claim 1, wherein steps(b)-(f) are performed using automated instrumentation.
 9. The method ofclaim 1, wherein step (c) is performed using a solid-phase extractiondevice comprising bonded silica sorbents, polymeric sorbents, orchelating agents.
 10. The method of claim 1, wherein prior to step (b),the sample is pre-treated with a solvent that facilitates disruption ofthe one or more microbes.
 11. The method of claim 1, wherein prior tostep (b) the sample is pre-treated with one or more organic solvents,provided that if only one organic solvent is used, the one organicsolvent is other than ethanol.
 12. A method for identifying one or moreunknown microbes, comprising: a) providing a sample suspected ofcontaining one or more unknown microbes; b) disrupting the one or moreunknown microbes present in the sample to provide a fluid extract; c)separating a soluble protein fraction from an insoluble protein fractionpresent in the fluid extract and preparing a solution containing intact,soluble proteins from the one or more unknown microbes; d) injecting thesolution into an ion source of a mass spectrometer system and ionizing astream or flow of the solution to form ionized proteins; e) analyzingthe ionized proteins with a mass analyzer of the mass spectrometersystem, wherein analyzing comprises: i) in a first mass spectrometrystep, acquiring one or more mass spectra representative of solubleproteins in the sample; ii) determining molecular weights for thesoluble proteins from the one or more mass spectra; iii) using thedetermined molecular weights to search a first database containingmolecular weights of known microbial proteins to identify, for a secondmass spectrometry step, a set of target proteins representative of asubset of candidate microbes; iv) in a second mass spectrometry step,selecting one or more precursor ions of the proteins from the one ormore ionized proteins of the sample and fragmenting the precursor ionsin the mass spectrometer by fragmentation means to produce a pluralityof product ions; v) using mass-to-charge ratios of the plurality ofproduct ions to search a second database containing molecular weights ofknown microbial proteins and at least one of amino acid sequence orpost-translational modification information of said known microbialproteins, wherein the subset of candidate microbes from the firstdatabase is used to limit a search of the second database; and f)automatically analyzing a sufficient number of proteins to identify atleast one of the one or more unknown microbes in said sample, whereinsteps (b)-(f) are completed in less than 25 minutes.
 13. The method ofclaim 12, wherein at least one of the first or the second massspectrometry step comprises selecting said one or more precursor ionsfrom a mass range window, and repeating said second mass spectrometrystep until a sufficient number of mass range windows have been coveredto identify at least one of the one or more microbes in said sample. 14.The method of claim 12, wherein step (c) is performed using asolid-phase extraction device comprising bonded silica sorbents,polymeric sorbents, or chelating agents.
 15. The method of claim 12,wherein prior to step (b), the sample is pre-treated with a solvent thatfacilitates disruption of the one or more microbes.
 16. The method ofclaim 12, wherein prior to step (b) the sample is pre-treated with oneor more organic solvents, provided that if only one organic solvent isused, the one organic solvent is other than ethanol.
 17. A method foridentifying and characterizing one or more unknown microbes, comprising:a) providing a first aliquot of a sample containing one or more unknownmicrobes; b) disrupting the one or more unknown microbes present in thefirst aliquot to provide a fluid extract; c) separating a first solubleprotein fraction from an insoluble protein fraction present in the fluidextract and preparing a first solution of soluble proteins; d) injectingthe first solution into an ion source of a mass spectrometer system andionizing the first solution to form ionized proteins; e) analyzing theionized proteins with a mass analyzer of the mass spectrometer system,wherein analyzing comprises: i) in a first mass spectrometry step,acquiring one or more first mass spectra representative of solubleproteins in the sample; ii) determining molecular weights for thesoluble proteins based upon the one or more first mass spectra; iii)using the determined molecular weights to perform a first search of afirst database containing molecular weights of known microbial proteinsto identify, for a second mass spectrometry step, a set of targetproteins representative of a first subset of candidate microbes; iv) inthe second mass spectrometry step, selecting one or more first precursorions of the one or more first ionized proteins and fragmenting in themass spectrometer the first precursor ions by fragmentation means toproduce a first plurality of first product ions; v) using mass-to-chargeratios of the first plurality of first product ions to perform a firstsearch of a second database containing molecular weights of knownmicrobial proteins and at least one of product ion m/z values, aminoacid sequence or post-translational modification information of saidknown microbial proteins, wherein the first subset of candidate from thefirst database is used to limit the first search of the second database;f) using information obtained in step (e) for one or more solubleproteins from each of the one or more unknown microbes in the sample toidentify at least one of the microbes to the genus species level,wherein steps (b)-(f) are completed in less than 25 minutes; g) usinginformation from step (f) to automatically select a second analyticalmethod from a list of pre-defined analytical methods, the second methodalso using mass spectrometry; h) providing a second aliquot of the fluidextract or of a second fluid extract of the same sample; i) separating asecond soluble protein fraction from an insoluble protein fractionpresent in the second aliquot and preparing a second solution of solubleproteins; j) subjecting the second solution to a separation to provide asubset of soluble proteins; k) injecting the second solution into an ionsource of a mass spectrometer system and ionizing a stream or flow ofthe subset of soluble proteins to form one or more second ionizedproteins; l) analyzing the one or more second ionized proteins with themass analyzer of the mass spectrometer system, wherein analyzingcomprises: i) in a third mass spectrometry step, acquiring one or moresecond mass spectra representative of one or more soluble proteins inthe subset of soluble proteins; ii) determining molecular weights forthe one or more soluble proteins in the subset of soluble proteins fromthe one or more second mass spectra; iii) performing a second search ofthe first database and selecting a second subset of the candidatemicrobes from the database on the basis of the determined molecularweights of the one or more soluble proteins in the subset of solubleproteins; iv) in a fourth mass spectrometry step, selecting one or moresecond precursor ions of the one or more second ionized proteins andfragmenting in the mass spectrometer the second precursor ions byfragmentation means to produce a second plurality of product ions; v)using mass-to-charge ratios of the second plurality of product ions toperform a second search of the second database, wherein the secondsubset of tentatively identified microbes from the first database isused to limit the second search of the second database; m) usinginformation obtained in step (l) to identify and, optionally toquantitate, one or more soluble proteins indicative of any of: strainand/or serovar identification, antibiotic resistance, antibioticsusceptibility, virulence or any combination thereof, wherein the methodincludes no chemical or enzymatic digestion of the proteins in the firstor second aliquot of the sample.
 18. The method of claim 17, wherein thesecond analytical method is performed using a second fluid extract ofthe same sample.
 19. The method of claim 17, wherein step (c) isperformed using a solid-phase extraction device comprising bonded silicasorbents, polymeric sorbents, or chelating agents.
 20. The method ofclaim 11, wherein the separation of step (j) comprises rapid,time-compressed chromatography which provides partial separation ofproteins in the second solution of the second soluble protein fraction.21. The method of claim 17, wherein prior to step (b), the sample ispre-treated with a solvent that facilitates disruption of the one ormore microbes.
 22. The method of claim 17, wherein prior to step (b) thesample is pre-treated with one or more organic solvents, provided thatif only one organic solvent is used, the one organic solvent is otherthan ethanol.
 23. The method of claim 17, wherein steps (a)-(m) areperformed using automated instrumentation.